Antibiotics exposure leads to gut microbiota dysbiosis, which increases the risk of anxiety and depression. However, the impact of ciprofloxacin and metronidazole exposure on chronic unpredictable mild stress-induced anxiety-like and depression-like behavior and underlying regulatory mechanism have not been well established. Here, chronic unpredictable mild stress model was established in adult male Sprague-Dawley rats. 16 S rRNA gene sequencing was used to decipher the gut microbiota. Enzyme-linked immunosorbent assay (ELIZA) was used to measure circulating cytokines in blood, gut barrier permeability biomarkers in feces, blood-brain barrier permeability biomarkers in brain. We found that antibiotics exposure significantly reduced the body weight, weight gain and liver health in chronic unpredictable mild stress treated rats. Behavioral testing suggested that antibiotics exposure reduced anxiety-like and depression-like behavior of rat. Antibiotics exposure possessed lower bacterial richness and diversity than that in the chronic unpredictable mild stress treated group. Compared with CUMS or CUMS-e group, higher abundances of Bacteroides, Lactobacillus, Lachnospiraceae and Akkermansia, lower abundances of S24-7, Blautia, Ruminocaceae, Ruminococcus and Prevotella were found in the gut microbiota from antibiotics exposure group. In addition, short-term antibiotics exposure increased the level of 5-hydroxytryptamine (5-HT) in brain. A significant correlation between certain bacteria and behavior of rats was observed, such as Roseburia. Our study uncovers the role for antibiotics in regulating chronic unpredictable mild stress-induced anxiety-like and depression-like behavior and suggest that short-term antibiotics exposure may be could reverse chronic unpredictable mild stress-induced anxiety-like and depression-like behavior.